The present invention relates to novel immortalized preosteoblast cell lines derived from the periosteal layer of bones which are capable of differentiating to osteoblasts. In particular, the present invention pertains to the use of such cell lines in assays for detecting substances controlling the differentiation of periosteally derived-preosteoblasts to osteoblasts and for detecting substances enabling improved bone formation, maintenance of bone mass, bone repair and for preventing the onset of osteoporosis.
In bone tissues, bones are constantly destructed, resorbed and created de novo by recurrent processes controlled on the cellular level by osteoblasts and osetoclasts, respectively. Osteoblasts are primarily involved in the process of bone formation, while the process of bone destruction and resorption of the bone material seems to be mediated by osteoclasts.
Bones are constituted of two major zones, an inner zone called “substantia spongosia”, and an outer zone, which is called “substantia corticalis”. Said outer zone harbors the Havers channels and the periost. The periost, the so called skin of the bones, covers the circumference of all bones with the proviso of those employed in joints. The periost primarily provides the bones with vessels, ascertains the attachment of tendons and harbors a plurality of nerves. Recently, it has been found that the periost also seems to participate in bone growth and furthermore in (bone) regenerating processes of an individual, such as bone repair after fracture.
The periost itself is composed of two major areas, the outer part, termed “stratum fibrosum”, which mainly contains connecting tissue, and the “Kambium” or “stratum osteogenicum”, which is adjacent to the bone itself and comprises a number of non-differentiated cells, such as e.g. preosteoblasts. It is presently hypothesized that these preosteoblasts, or the cells derived therefrom, seem to participate in bone fracture repair and/or other reconstitutive processes. These preosteoblasts are, however, seemingly different from preosteoblasts found in the “substantia spongosia”, since said cells are deemed to lack the capability of differentiating into adipocytes and are supposed not to support osteoclast differentiation.
During ageing an individual is subject to a gradual loss in bone mass involving a widening of the Havers channels in the substantia corticalis and a reduction in mass of the substantia spongosia. This phenomenon is mainly due, on the cellular level, by bone resorption of osteoclasts exceeding bone formation by osteoblasts, which condition is termed uncoupling. In case the uncoupling persists for a longer period of time, more and more of the bone's material gets destructed/resorbed finally generating a disease termed osteoporosis. Osteoporosis causes pain in the bone and renders the bone fragile, eventually leading to fracture thereof and lumbago.
In the past osteoporosis has been treated by various regimens, involving increasing the calcium intake, light exercises, sun tan or administering compounds increasing the activity of osteoblasts present in the “substantia spongosia”. In this respect, the U.S. Pat. No. 5,002,968 discloses an organogermanium compound for activating proliferation of osteoblasts so as to stimulate bone formation and to balance the surpassing activity of osteoclasts. Further, in EP 0 725 080 a novel protein, the basic osteoblast growth factor II (bOGF-II) is disclosed, which is capable to stimulate osteoblast growth.
However, quite recently it was found that osteoblasts not only perform a task in ossification but also seem to play a role as a control center for the bone reformation phenomenon that is closely related to the differentiation and activation of osteoclasts, the cells mediating bone degradation. In view of this, the mere activation of existing osteoblasts for the promotion and maintenance of bone mass is now rather doubted to be efficient.
Concurrently, it has been found that at the same time as bone loss is occurring within the bones, bone is being added to the periostal surface, yet more slowly than during growth, indicating this process is contravening the destabilization of bones effected by bone degradation.
It would therefore be interesting to evaluate and study the osteoblasts present in the periost and their action on bone repair and bone build up so as to provide substances that influence said processes in the periost. In order to provide such compounds, effective means for elucidating the effect thereof on cells involved in bone metabolism are in need.
As is acknowledged in the art the best means for performing such experiments are cells involved in said processes. However, cells directly obtained from a donor, the so called primary cells, have a limited proliferative lifespan only, which restricts the use thereof for in vitro studies. In addition, not all type of cells may be isolated and cultured. This applies in particular to precursor cells that often may not be isolated in sufficient quantity so that experiments on said cells may be carried out.
In the past, though some cells proved to be resistant to a manipulation of this kind, the proliferation of certain cells, as e.g. intestinal or corneal cells, could be extended by infecting cells of a primary cell culture with oncogenes, such as the siminian virus 40 large T antigen (SV40 T antigen). The SV40 large T-antigen is known to effect inactivation of proteins playing a key role in cell cycle progressing, in particular the p53 and the retinoblastoma (pRb) protein. However, though the (over-)expression of such oncogenes in human primary cells could extend the proliferation of said cells for a limited number of cell divisions, said cells eventually stop growing at a stage called “crisis”. During this stage, which normally occurs at about passage 10 to 20 the cells remain viable in a state named “senescence” or simply die. In rare cases, some cells may escape from this state and start to proliferate again. This capability to start proliferation de novo is accounted for by genomic reorganisation through additional epigenic events taking place in the cell. Yet, these substantial genome modifications most often result in the cells loosing the initial characteristic of the primary cells they are derived from.
Consequently, a problem underlying the present invention is to provide a means for further determining substances influencing bone formation and various effects thereof on bone metabolism.